Intra-articular injection of gold micro-particles with hyaluronic acid for painful knee osteoarthritis.

BACKGROUND: Recently, in an open pilot study, we found up to two years, a potential pain-relieving effect of intra-articular gold micro-particles using the patient's synovial fluid for patients with knee osteoarthritis (KOA). During the study the excluded group of patients, due to multisite pain, co-morbidities, and other exclusion criteria., received intra-articular gold micro-particles using hyaluronic acid,. We aimed to identify if pre-treatment characteristics influence the global outcome two years after intra-articular treatment for painful KOA with gold microparticles using hyaluronic acid. METHODS: Using hyaluronic acid as the carrier, 136 patients with KOA received intraarticular injections with 20 mg gold microparticles (72.000 particles, 20-40 µm in diameter). In the analysis, we included the Global Rating of Change Scale, Pain Detect Questionnaire (PDQ), Body Mass Index (BMI), and Kellgren & Lawrence score at the inclusion, Western Ontario, and McMaster Universities Osteoarthritis Index (WOMAC) sub-scores for pain, stiffness, and function at inclusion and two years. RESULTS: On the Global Rating Change Scale, 69.1% of patients reported a positive effect, 28.7% no effect, and 2.2% worse. PDQ and the three WOMAC subscores all improved at two years of follow-up. PDQ ≥ 13 (P = 0.028), BMI (P = 0.022) and Kellgren & Lawrence grade 4 (P = 0.028) at inclusion reduced the effect with a minor odds ratio compared to the baseline effect of treatment (P = 0.025). WOMAC subscores at inclusion did not influence the outcome (P > 0.5). CONCLUSIONS: Severe osteoarthritis, obesity, and neuropathic pain, reduced the effect of intra-articular gold microparticles for knee OA. TRIAL REGISTRATION: The study followed the principles of the Declaration of Helsinki and was approved by the local ethics committee of the North Denmark Region by 27/07/2016 (N-20,160,045). The regional data protection agency approved the project by 06/07/2016 (2008-58-0028, ID 2016 - 116) and registered in ClinicalTrial.Gov by 04/01/2018 (NCT03389906).

New Enhancing MRI Lesions Associate with IL-17, Neutrophil Degranulation and Integrin Microparticles: Multi-Omics Combined with Frequent MRI in Multiple Sclerosis.

BACKGROUND: Blood-barrier (BBB) breakdown and active inflammation are hallmarks of relapsing multiple sclerosis (RMS), but the molecular events contributing to the development of new lesions are not well explored. Leaky endothelial junctions are associated with increased production of endothelial-derived extracellular microvesicles (EVs) and result in the entry of circulating immune cells into the brain. MRI with intravenous gadolinium (Gd) can visualize acute blood-barrier disruption as the initial event of the evolution of new lesions. METHODS: Here, weekly MRI with Gd was combined with proteomics, multiplex immunoassay, and endothelial stress-optimized EV array to identify early markers related to BBB disruption. Five patients with RMS with no disease-modifying treatment were monitored weekly using high-resolution 3T MRI scanning with intravenous gadolinium (Gd) for 8 weeks. Patients were then divided into three groups (low, medium, or high MRI activity) defined by the number of new, total, and maximally enhancing Gd-enhancing lesions and the number of new FLAIR lesions. Plasma samples taken at each MRI were analyzed for protein biomarkers of inflammation by quantitative proteomics, and cytokines using multiplex immunoassays. EVs were characterized with an optimized endothelial stress EV array based on exosome surface protein markers for the detection of soluble secreted EVs. RESULTS: Proteomics analysis of plasma yielded quantitative information on 208 proteins at each patient time point (n = 40). We observed the highest number of unique dysregulated proteins (DEPs) and the highest functional enrichment in the low vs. high MRI activity comparison. Complement activation and complement/coagulation cascade were also strongly overrepresented in the low vs. high MRI activity comparison. Activation of the alternative complement pathway, pathways of blood coagulation, extracellular matrix organization, and the regulation of TLR and IGF transport were unique for the low vs. high MRI activity comparison as well, with these pathways being overrepresented in the patient with high MRI activity. Principal component analysis indicated the individuality of plasma profiles in patients. IL-17 was upregulated at all time points during 8 weeks in patients with high vs. low MRI activity. Hierarchical clustering of soluble markers in the plasma indicated that all four MRI outcomes clustered together with IL-17, IL-12p70, and IL-1ß. MRI outcomes also showed clustering with EV markers CD62E/P, MIC A/B, ICAM-1, and CD42A. The combined cluster of these cytokines, EV markers, and MRI outcomes clustered also with IL-12p40 and IL-7. All four MRI outcomes correlated positively with levels of IL-17 (p < 0.001, respectively), and EV-ICAM-1 (p < 0.0003, respectively). IL-1ß levels positively correlated with the number of new Gd-enhancing lesions (p < 0.01), new FLAIR lesions (p < 0.001), and total number of Gd-enhancing lesions (p < 0.05). IL-6 levels positively correlated with the number of new FLAIR lesions (p < 0.05). Random Forests and linear mixed models identified IL-17, CCL17/TARC, CCL3/MIP-1α, and TNF-α as composite biomarkers predicting new lesion evolution. CONCLUSIONS: Combination of serial frequent MRI with proteome, neuroinflammation markers, and protein array data of EVs enabled assessment of temporal changes in inflammation and endothelial dysfunction in RMS related to the evolution of new and enhancing lesions. Particularly, the Th17 pathway and IL-1ß clustered and correlated with new lesions and Gd enhancement, indicating their importance in BBB disruption and initiating acute brain inflammation in MS. In addition to the Th17 pathway, abundant protein changes between MRI activity groups suggested the role of EVs and the coagulation system along with innate immune responses including acute phase proteins, complement components, and neutrophil degranulation.

Impaired Abcb1a function and red meat in a translational colitis mouse model induces inflammation and alters microbiota composition.

Inflammatory Bowel Disease (IBD) affects approximately 0.3% of the global population, with incidence rates rising dramatically worldwide. Emerging evidence points to an interplay between exposome factors such as diet and gut microbiota, host genetics, and the immune system as crucial elements in IBD development. ATP-binding cassette (ABC) transporters, including human p-glycoprotein encoded by the Abcb1 gene, influence intestinal inflammation, and their expression may interact with environmental factors such as diet and gut microbes. Our study aimed to examine the impact of protein sources on a genetic colitis mouse model. Methods: Abcb1a-deficient colitis mice were fed either casein or red meat-supplemented diets to investigate potential colitis-aggravating components in red meat and their effects on host-microbiota interactions. We conducted deep label free quantitative proteomic inflammation profiling of gastrointestinal tissue (colon, ileum) and urine, and determined the overall microbiome in feces using 16S rRNA gene sequencing. Microbiota shifts by diet and protein transporter impairment were addressed by multivariate statistical analysis. Colon and systemic gut inflammation were validated through histology and immune assays, respectively. Results: A quantitative discovery based proteomic analysis of intestinal tissue and urine revealed associations between ileum and urine proteomes in relation to Abcb1a deficiency. The absence of Abcb1a efflux pump function and diet-induced intestinal inflammation impacted multiple systemic immune processes, including extensive neutrophil extracellular trap (NET) components observed in relation to neutrophil degranulation throughout the gastrointestinal tract. The colitis model's microbiome differed significantly from that of wild-type mice, indicating the substantial influence of efflux transporter deficiency on microbiota. Conclusion: The proteomic and microbiota analyzes of a well-established murine model enabled the correlation of gastrointestinal interactions not readily identifiable in human cohorts. Insights into dysregulated biological pathways in this disease model might offer translational biomarkers based on NETs and improved understanding of IBD pathogenesis in human patients. Our findings demonstrate that drug transporter deficiency induces substantial changes in the microbiota, leading to increased levels of IBD-associated strains and resulting in intestinal inflammation. GRAPHICAL ABSTRACT.

Gold micro-particles for knee osteoarthritis.

BACKGROUND: This exploratory study investigates if intra-articular injected gold microparticles in knee osteoarthritis (KOA) reduce immunomodulatory-based pain via proteomic changes in the synovial fluid (SF) and serum. METHODS: Thirty patients with moderate KOA were included. Intraarticular injections with 20 mg gold microparticles (72.000 particles, 20-40 µm in diameter) using the patient's synovial fluid (SF) as carrier were performed. Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) subscores for pain, stiffness, and function were assessed at inclusion, 8 weeks and 2 years The PainDetect questionnaire, pain pressure threshold (PPT), temporal summation (TS), and conditioned pain modulation (CPM), and pain diary were assessed at inclusion and 8 weeks. Proteome analysis was performed on SF and blood samples before and after 8 weeks of treatment. RESULTS: A decrease in WOMAC scores (pain (p = 0.0001), stiffness (p = 0.0088), activity (p = 0.0001)), PainDetect (p = 0.0002) and increase in PPT (p = 0.001) and CPM (p = 0.021) and a decrease in TS (p = 0.03) were found after 8 weeks compared to inclusion assessments. At 2 years follow-up compared to baseline there was a decrease in WOMAC scores (pain (p = 0.0001), stiffness (p = 0.007), activity (p = 0.0001)) and PainDetect (p = 0.0001). In SF, 28 different proteins were downregulated and 11 upregulated (p < 0.05) mainly associated immune response. Similarly, 31 proteins were downregulated and 1 upregulated in serum (p < 0.05) reflecting key immune response and anatomical structure development processes. No adverse effects related to the treatment were recorded. CONCLUSIONS: Gold microparticles injected intra-articular in KOA joints may provide pain relief and an inflammatory modulatory effect based on proteome changes found in SF and serum. A randomized, controlled, double-blind study is needed to infer a conclusion. SIGNIFICANCE: This study indicates that intra-articular gold may provide advantages in clinical practice for managing knee osteoarthritic pain. The use of intraarticular gold can add new knowledge to the treatment of inflammation and pain.

Protein array-based companion diagnostics in precision medicine.

INTRODUCTION: The development of companion diagnostics (CDx) will increase efficacy and cost-benefit markedly, compared to the currently prevailing trial-and-error approach for treatment. Recent improvements in high-throughput protein technology have resulted in large amounts of predictive biomarkers that are potentially useful components of future CDx assays. Current high multiplex protein arrays are suitable for discovery-based approaches, while low-density and more simple arrays are suitable for use in point-of-care facilities. AREA COVERED: This review discusses the technical platforms available for protein array focused CDx, explains the technical details of the platforms and provide examples of clinical use, ranging from multiplex arrays to low-density clinically applicable arrays. We thereafter highlight recent predictive biomarkers within different disease areas, such as oncology and autoimmune diseases. Lastly, we discuss some of the challenges connected to the implementation of CDx assays as point-of-care tests. EXPERT OPINION: Recent advances in the field of protein arrays have enabled high-density arrays permitting large biomarker discovery studies, which are beneficial for future CDx assays. The density of protein arrays range from a single protein to proteome-wide arrays, allowing the discovery of protein signatures that may correlate with drug response. Protein arrays will undoubtedly play a key role in future CDx assays.

Absence of miRNA-146a Differentially Alters Microglia Function and Proteome.

Background: MiR-146a is an important regulator of innate inflammatory responses and is also implicated in cell death and survival. Methods: By sorting CNS resident cells, microglia were the main cellular source of miR-146a. Therefore, we investigated microglia function and phenotype in miR-146a knock-out (KO) mice, analyzed the proteome of KO and wild-type (WT) microglia by LC-MS/MS, and examined miR-146a expression in different brain lesions of patients with multiple sclerosis (MS). Results: When stimulated with LPS or myelin in vitro, microglia from KO mice expressed higher levels of IL-1ß, TNF, IL-6, IL-10, CCL3, and CCL2 compared to WT. Stimulation increased migration and phagocytosis of WT but not KO microglia. CD11c+ microglia were induced by cuprizone (CPZ) in the WT mice but less in the KO. The proteome of ex vivo microglia was not different in miR-146a KO compared to WT mice, but CPZ treatment induced differential and reduced protein responses in the KO: GOT1, COX5b, CRYL1, and cystatin-C were specifically changed in KO microglia. We explored discriminative features of microglia proteomes: sparse Partial Least Squares-Discriminant Analysis showed the best discrimination when control and CPZ-treated conditions were compared. Cluster of ten proteins separated WT and miR-146a KO microglia after CPZ: among them were sensomes allowing to perceive the environment, Atp1a3 that belongs to the signature of CD11c+ microglia, and proteins related to inflammatory responses (S100A9, Ppm1g). Finally, we examined the expression of miR-146a and its validated target genes in different brain lesions of MS patients. MiR-146 was upregulated in all lesion types, and the highest expression was in active lesions. Nineteen of 88 validated target genes were significantly changed in active lesions, while none were changed in NAWM. Conclusion: Our data indicated that microglia is the major source of miR-146a in the CNS. The absence of miR-146a differentially affected microglia function and proteome, and miR-146a may play an important role in gene regulation of active MS lesions.